Hydrolysable Polymeric FMOC-Linker

ABSTRACT

The invention relates to Fmoc (9-fluorenyl-methoxycarbonyl)-based polymeric conjugates. These conjugates are useful for extending the in-vivo circulation of protein and peptide drugs.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority to U.S. provisionalapplication Ser. No. 60/937,169, filed Jun. 26, 2007 and to U.S.provisional application Ser. No. 61/123,263, filed Apr. 7, 2008, whichare both incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the preparation of a hydrolysablelinker, which is bound to at least one semi-synthetic polymer. Thesehydrolysable linker are useful for extending the in-vivo circulation ofprotein and peptide drugs.

BACKGROUND OF THE INVENTION

Most protein or peptide drugs are short-lived and have often a shortcirculatory half-life in vivo. Considering that protein or peptide drugsare not absorbed orally, prolonged maintenance of therapeutically activedrugs in the circulation is a desirable feature of obvious clinicalimportance.

An attractive strategy for improving clinical properties of protein orpeptide drugs is a modification of the drugs with polymers e.g.polyalkylene-oxides (Roberts et al., Advan Drug Rev. 54, 459-476 (2002))or polysaccharides like polysialic acid (Fernandes et al., BiochimBiophys Acta 1341, 26-34 (1997)), dextranes or hydroxyl alkyl starch.(All documents cited in the specification are incorporated byreference.)

The modification with poly(ethylene glycol) (PEG) has been known for awhile. However, modification of proteins with PEG often leads toreduction of the activity of the protein.

Polysialic acid (PSA), also known as colominic acid (CA), is a naturaloccurring polysaccharide. It is a homopolymer of N-acetylneuraminic acidwith α(2→8) ketosidic linkage and contains vicinal diol groups at itsnon-reducing end. PSA is negatively charged and is a natural constituentof the human body. It can easily be produced from bacteria in largequantities with pre-determined physical characteristics (U.S. Pat. No.5,846,951). Being chemically and immunologically identical to polysialicacid in the human body, bacterial polysialic acid is non-immunogeniceven when coupled to proteins. Unlike other polymers (e.g.; PEG),polysialic acid is biodegradable.

However, to date no therapeutic compound comprising a polypeptideconjugated to an acidic monosaccharide such as PSA is commerciallyavailable.

Short PSA polymeric chains with only 1-4 sialic acid units have alsobeen synthesized (Kang et al., Chem. Commun., 227-228 (2000); Ress etal., Current Organic Synthesis 1, 31-46 (2004)).

Several hydrolysable or degradable linkers comprising PEG moieties havebeen suggested.

U.S. Pat. No. 6,515,100, describes PEG and related polymer derivatives,having weak, hydrolytically unstable linkages

U.S. Pat. No. 7,122,189 describes releasable PEG-linkers based onbis-N-2-hydroxyethyl glycine groups (bicine).

WO 04/089280 and WO 06/138572 describe hydrolysable fluorene-based PEGconstructs.

After conjugation of these linkers to protein drugs, the protein-polymerconjugate can be regarded as a prodrug and the activity of the proteincan be released from the conjugate via a controlled release mechanism.Using this concept improved pharmacokinetic properties of the drug canbe obtained (Zhao et al., Bioconjugate Chem. 17, 341-351 (2006)).

SUMMARY OF THE INVENTION

The present invention provides a hydrolysable linker, which is bound toat least one semi-synthetic biopolymer, wherein the hydrolysable linkeris conjugated to a protein or peptide drug in order to improve itsin-vivo properties such as the in-vivo circulation.

The present invention provides a compound of the general formula 1:

wherein Z a leaving group and at least one of position 1, 2, 3, 4, 5, 6,7 or 8 is bound to radical Y.

Y is a radical containing a semi-synthetic biopolymer, which is bound toa N-succinimidyl moiety.

In addition to being bound to radical Y the compound of formula 1 mayoptionally be bound to radical X in at least one of the availableposition 1, 2, 3, 4, 5, 6, 7 or 8.

X is —SO₃—R³.

R³ is selected from the group consisting of hydrogen, (C₁-C₈)-alkyl and(C₁-C₈)-alkyl-R⁴.

R⁴ is a polymer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the in-vitro hydrolysis of a FVIIa-PSA conjugate at pH 8.3.The release of the FVIIa activity was measured with the Staclot-assay(Diagnostica Stago, Asnières, France).

FIG. 2 shows the shows the in-vitro hydrolysis of a FVIIa-trimer PSAconjugate at pH 8.3. The release of the FVIIa activity was measured withthe Staclot-assay (Diagnostica Stago, Asnières, France).

FIG. 3 shows FVIIa activity in plasma measured with a clotting assay(Staclot, Diagnostica Stago, Asnières, France). For FVIIa clottingactivity the dose adjusted area under curve (AUC) was 0.014 forunmodified rFVIIa and increased to 0.015 for rFVIIa-conjugate(0-infinity). The terminal half-life increased from 2.3 to 4.4 hours andthe mean residence time (MRT) from 1.4 to 2.4 hours.

FIG. 4 shows the determination of the FVIIa antigen by ELISA with apolyclonal anti-human FVII antibody. For the antigen the dose adjustedAUC (0-infinity) increased from 0.010 (unmodified rFVIIa) to 0.014(rFVIIa-conjugate), the terminal half life increased from 1.4 to 2.3hours and the MRT from 1.5 to 2.2 hours.

FIG. 5 shows FVIIa activity in plasma, measured with a clotting assay(Staclot, Diagnostica Stago, Asnières, France). The pharmacokinetic ofrFVIIa-trimer-PSA conjugates is improved (-◯-) compared to native rFVIIa(--Δ--).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a hydrolysable linker, which is bound toat least one semi-synthetic biopolymer, wherein the hydrolysable linkercan be further conjugated to a protein or peptide drug in order toimprove their in-vivo properties such as in-vivo circulation. Theactivity of the protein or peptide drug can be released from theconjugate via a controlled release mechanism.

The following paragraphs provide general definitions and definition ofvarious chemical moieties that make up the compounds according to theinvention and are intended to apply uniformly through-out thespecification and claims unless an otherwise expressly set outdefinition provides a broader definition.

“C₁-C₈-alkyl” refers to monovalent alkyl groups having 1 to 8 carbonatoms. This term is exemplified by groups such as methyl, ethyl, propyl,butyl, hexyl and the like. Linear and branched alkyls are included.

“Leaving groups” refers to groups, which are capable of reacting with anucleophile present on the protein or peptide drug that forms theconjugate. This term is exemplified by groups such asN-hydroxysuccimimidyl, N-hydroxybenzotriazolyl, halogen,N-hydroxyphthalimidyl, p-nitrophenoxy, imidazolyl, thiazolidinyl thione,O-acyl ureas or other suitable leaving groups will be apparent to thoseof ordinary skill. For the purpose of the present invention, the proteinor peptide drug thus contains one or more groups for displacement, suchas an amine. Protein or peptide drug are plasma proteins or bloodcoagulation factors such as FVIII, VWF, FVIIa and FIX.

A “semi-synthetic biopolymer” refers to a manufactured organic polymer,which is based on a naturally occurring polymer. A semi-syntheticbiopolymer may also be functionalized by reactive groups in order toconjugate these functionalized semi-synthetic biopolymers to othercompounds. This term “semi-synthetic biopolymer” is exemplified bylinear or branched polymers such as carbohydrates, specifically such aspolysaccharides. Examples of polysaccharides are PSA (polysialic acid)and HAS (hydroxyalkylstarch).

“Hydrolysable” linker refers to a linker system, in which the protein isreleased in native form. The protein is released and the linker is splitoff completely. Synonyms for hydrolysable are “degradable” or“releasable” linkers.

The present invention provides a compound of the general formula 1:

wherein Z a leaving group and at least one of position 1, 2, 3, 4, 5, 6,7 or 8 is bound to radical Y.

Y is a radical containing a semi-synthetic biopolymer, which is bound toa N-succinimidyl moiety.

In addition to being bound to radical Y the compound of formula 1 mayoptionally be bound to radical X in at least one of the availableposition 1, 2, 3, 4, 5, 6, 7 or 8.

X is —SO₃—R³.

R³ is selected from the group consisting of hydrogen, (C₁-C₈)-alkyl and(C₁-C₈)-alkyl-R⁴.

R⁴ is a polymer. Examples are hydrophilic polymers such as poly(ethyleneglycol) (PEG).

In one embodiment, the invention relates to a compound of formula 1,wherein Z is an N-succinimidyl ester and at least one of position 1, 2,3, 4, 5, 6, 7 or 8 is bound to radical Y, wherein Y is:

wherein POLYMER is a semi-synthetic biopolymer, preferably with amolecular weight of 1,000 Da to 300,000 Da.

In one embodiment the molecular weight is 5,000-25,000, preferably5,000-10,000.

In another embodiment said semi-synthetic biopolymer is a carbohydrate,preferably a polysaccharide, preferably comprising at least 3 units of amonosaccharide.

In one embodiment said polysaccharide comprises between 2-200 units,preferably between 10-100 units of a monosaccharide.

In one embodiment the semi-synthetic biopolymer is a PSA derivative.

In another embodiment the semi-synthetic biopolymer is bound to thesuccinimidyl moiety via a thioether linkage.

R¹ is at each occurrence independently a (C₁-C₈)-alkyl.

In one embodiment R¹ is at each occurrence independently selected fromthe group consisting of methyl, ethyl, propyl, butyl, and hexyl.

R² is independently selected from the group consisting of —C(O)NR—,—C(O)NR—(C₁-C₈)-alkyl-NR—, —NRC(O)— and —NRC(O)—(C₁-C₈)-alkyl-NR,wherein R is independently either hydrogen or (C₁-C₈)-alkyl.

In one embodiment R² is —C(O)NH—.

In another embodiment R² is —NHC(O)—.

In one embodiment the compound of formula 1 is bound to radical Y in atleast one of position 1, 2, 3 or 4.

In another embodiment the compound of formula 1, is bound to radical Yin at least one of position 1, 2, 3, or 4 and is further bound toradical X in at least one of position 5, 6, 7, or 8.

In another embodiment the compound of formula 1, is bound to at leastone radical Y in at least one of position 2 or 3 is further bound toradical X in at least one of position 7 or 8.

In another embodiment the compound of formula 1 is bound to radical Y inpositions 2 and 7.

In another embodiment the compound of formula 1 is bound to radical Yand radical X in positions 2 and 7, respectively.

In another embodiment the compound of formula 1 is:

In a further embodiment, the invention relates to the preparation of acompound of formula 1.

Tsubery et al., J Biol. Chem. 279, 38118-38124 (2004) described thesynthesis of a hydrolysable PEG-linker for derivatization of proteinsbased on the Fmoc (9-fluorenyl-methoxycarbonyl)-moiety. The synthesis ofMAL-FMS-OSU (9-Hydroxymethyl-2-(amino-3-maleimido-propionate)-7-sulfofluorene N-hydroxysuccinimidyl carbonate) is described. The syntheticscheme below illustrates the synthetic steps for the preparation of acompound of formula 1 as an example, starting from a MAL-FMS-OSUderivative.

wherein

POLYMER is a semi-synthetic biopolymer;

R¹ is at each occurrence independently a (C₁-C₈)-alkyl;

R² is independently selected from the group consisting of —C(O)NR—,—C(O)NR—(C₁-C₈)-alkyl-NR—, —NRC(O)— and —NRC(O)—(C₁-C₈)-alkyl-NR,wherein R is independently either hydrogen or C₁-C₈-alkyl.

R is independently either hydrogen or (C₁-C₈)-alkyl;

X is —SO₃—R³;

R³ is independently selected from the group consisting of hydrogen,(C₁-C₈)-alkyl and (C₁-C₈)-alkyl-R⁴;

R⁴ is a polymer;

n is an integer selected from 0, 1, 2, 3, or 4; and

m is an integer selected from 1, 2, 3, or 4.

HS-POLYMER is a thiol-derivatized semi-synthetic biopolymer, such as

A compound of formula 2 can be easily reacted with a protein or peptidedrug containing one or more groups for displacement, such as amines.Preferred protein or peptide drug are blood coagulation factors such asFVIII, VWF, FVIIa, FIX.

Protein and peptide drugs modified according to the above protocol havea significantly increased in-vivo circulation. The hydrolysability ofthe linker allows that the activity can be regained after hydrolysis, byrelease of the protein in its native form. An example is shown in FIGS.1 and 2. The restoration of the biological activity of a proteinconjugate is shown in FIGS. 3 and 4.

The present invention is illustrated by the following examples withoutbeing limited thereto.

EXAMPLES Example 1 Preparation of PSA Containing Terminal SH Groups

Polysialic acid (Sigma) was oxidized with NaIO₄ (Fernandes et al.,Biochim Biophys Acta 1341, 26-34 (1997)), and a terminal aldehyde groupwas formed. Then a reductive amination step with NH₄Cl was carried outas described in WO 05/016973 and the Schiff Base was reduced withNaCNBH₃ to form PSA-NH₂ containing a terminal amino group. Subsequentlya reaction with 2-iminothiolane (Pierce 26101) was performed accordingto the instruction leaflet of the manufacturer to prepare a modified PSAcontaining a terminal SH group. The molarity of the generated SH-groupswas determined using Ellmans reagent. In addition the same procedure wasused to introduce a SH-group in a N-Acetylneuramic acid trimer, whichwas obtained from TimTec, LLC, Newark, USA.

Example 2 Conjugation of rFVIIa with PSA Using the MAL-FMS-OSU Linker

To 15 ml of a solution of rFVIIa (0.7 mg/ml) in 50 mM phosphate bufferpH 7.2 the bifunctional linker MAL-FMS-OSU (prepared as outlined byTsubery et al., J Biol. Chem. 279, 38118-38124 (2004)) was added(concentration: 0.5 mg/mg protein) and incubated at R.T. for 30 min.Then derivatized PSA containing a terminal SH group was preparedaccording to Example 1. The PSA derivative was added to the mixture(concentration: 10 mg PSA-SH/mg protein) and incubated for additional 2hours. Then the reaction was stopped by adding an aqueous solution of0.1 M glycine (final concentration 10 mM) and 5 mM cysteine (endconcentration 0.5 mM).

The free reagents were separated from the rFVIIa-PSA conjugate by ionexchange chromatography using a QHyperD F 50 μm resin (BioSepra) and aPharmacia XK-10 column (Pharmacia XK 10; h=10 cm). The PSA-rFVIIacontaining solution was applied to the column, which was subsequentlywashed with 10 CV equilibration buffer (20 mM sodium citrate, 20 mMNaCl, pH 6.5). Then the polysialylated rFVIIa was eluted with elutionbuffer (20 mM sodium citrate, 500 mM NaCl, pH 6.1). The eluate contained0.06 mg/ml protein, the evidence of bound PSA in the conjugate wasproven by the resorcinol assay (Svennerholm; Biochim Biophys Acta 24:604-11 (1957)). For release of the activity of rFVIIa in the conjugate450 μl of the eluate was added to 50 μl 1 M TRIS-buffer pH 8.3 and therelease of the FVIIa activity was measured (Staclot, Diagnostica Stago,Asnières, France). The results are illustrated in FIG. 2.

Example 3 Conjugation of rFVLLa with Trimer PSA Using the MAL-FMS-OSULinker

To 15 ml of a solution of rFVIIa (0.7 mg/ml) in 50 mM phosphate bufferpH 7.2 the bifunctional linker MAL-FMS-OSU (prepared as outlined byTsubery et al., J Biol. Chem. 279, 38118-38124 (2004)) was added(concentration: 0.07 mg/mg protein) and incubated at R.T. for 30 min.Then trimer PSA (TimTec, LLC, Newark, USA) was derivatized as describedin Example 1 to introduce a free SH-group. The trimer PSA-SH derivativewas added to the mixture (concentration: 0.43 mg trimer PSA-SH/mgprotein) and incubated for additional 2 hours. Then the reaction wasstopped by adding an aqueous solution of 0.1 M glycine (finalconcentration 10 mM) and 5 mM cysteine (end concentration 0.5 mM). Thefree reagents were separated from the rFVIIa-PSA conjugate by ionexchange chromatography using a QHyperD F 50 μm resin (BioSepra) and aPharmacia XK-10 column (Pharmacia XK 10; h=10 cm). The PSA-rFVIIacontaining solution was applied to the column, which was subsequentlywashed with 10 CV equilibration buffer (20 mM sodium citrate, 20 mMNaCl, pH 6.5). Then the polysialylated rFVIIa was eluted with elutionbuffer (20 mM sodium citrate, 500 mM NaCl, pH 6.1). The eluate contained0.06 mg/ml protein, the evidence of bound PSA in the conjugate wasproven by the resorcinol assay (Svennerholm et al., Biochim Biophys Acta24, 604-11 (1957)). For release of the activity of rFVIIa in theconjugate 450 μl of the eluate was added to 50 μl 1 M TRIS-buffer pH 8.3and the release of the FVIIa activity was measured (Staclot, DiagnosticaStago, Asnières, France). The results are illustrated in FIG. 1.

Example 4 Conjugation of Human Serum Albumin with PSA Using theMAL-FMS-OSU Linker

Human Serum Albumin (HSA) is incubated with the bifunctional linkerMal-FMSOSU linker (prepared as outlined by Tsubery et al., J Biol. Chem.279, 38118-38124 (2004)) in 25 mM sodium acetate buffer, pH 6.2 for 1hour. Then the excess linker is separated by gelfiltration usingSephadex G-25 (GE-Healthcare) using the same buffer system The proteincontaining fractions are collected and PSA-SH (prepared according toExample 1) is added. The mixture is incubated for 2 hours at R.T. Thenthe conjugate is purified by anion-exchange chromatography usingDEAE-Sepharose FF (GE Healthcare). The Protein-PSA conjugate is elutedwith 25 mM sodium acetate buffer pH 4.5. The conjugate containingfractions are pooled and concentrated by ultrafiltration using a 10Kmembrane. Then the solution is diafiltrated against 25 mM sodium acetatebuffer, pH 6.2.

Example 5 Pharmacokinetic of rFVIIa-PSA-Conjugate in Normal Rats

A rFVIIa-PSA conjugate was prepared according to Example 2 using aconcentration of MAL-FMS-OSU of 0.05 mg/mg protein. 8 normal rats (4male, 4 female) were anaesthetized and the rFVIIa-PSA-conjugate inbuffer (1.3 g/L glycylglycine, 3 g/L sodium chloride, 30 g/L mannitol,1.5 g/L CaCl₂×2H₂O, 0.1 g/L Tween 80, pH 5.5) was applied by intravenousinjection into the tail vein in a volume dose of 10 ml per kg (1200 μgprotein/kg). Unmodified rFVIIa in a dose of 1200 μg protein/kg was usedas control in 8 normal rats (4 male, 4 female). Blood samples were takenfrom the tail artery 5 minutes, 1 hour, 2, 4, 7, 10 and 24 hours aftersubstance application and citrated plasma was prepared and frozen forfurther analysis.

FVIIa activity in plasma was measured with a clotting assay (Staclot,Diagnostica Stago, Asnières, France), FVII antigen was determined withan ELISA (polyclonal anti-human FVII antibody). The results wereevaluated statistically. For FVIIa clotting activity the dose adjustedarea under curve (AUC) was 0.014 for unmodified rFVIIa and increased to0.015 for rFVIIa-conjugate (0-infinity). The terminal half-lifeincreased from 2.3 to 4.4 hours and the mean residence time (MRT) from1.4 to 2.4 hours. For the antigen the dose adjusted AUC (0-infinity)increased from 0.010 (unmodified rFVIIa) to 0.014 (rFVIIa-conjugate),the terminal half life increased from 1.4 to 2.3 hours and the MRT from1.5 to 2.2 hours. All calculations were carried out by use of astatistical program (program R: A language and environment forstatistical computing. R Foundation for Statistical Computing, Vienna,Austria, ISBN 3-900051-07-0, URL http://www.R-project.org). Thepharmacokinetic results are illustrated in FIGS. 3 and 4.

Example 6 Pharmacokinetic of rFVIIa-Trimer-PSA-Conjugate in Normal Rats

rFVIIa-trimer-PSA conjugate was prepared according to Example 3 using aMAL-FMS-OSU concentration of 0.05 mg/mg protein. 6 normal rats (3 male,3 female) were anaesthetized and the rFVIIa-trimer-PSA-conjugate inbuffer (1.3 g/L glycylglycine, 3 g/L sodium chloride, 30 g/L mannitol,1.5 g/L CaCl₂×2H₂O, 0.1 g/L Tween 80, pH 5.5) was applied by intravenousinjection into the tail vein in a volume dose of 10 ml per kg (1200 μgprotein/kg). Unmodified rFVIIa in a dose of 1200 μg protein/kg was usedas a control in 6 normal rats (3 male, 3 female). Blood samples weretaken from the tail artery 5 minutes, 1 hour, 2, 4, 7, 10 and 24 hoursafter substance application and citrated plasma was prepared and frozenfor further analysis.

FVIIa activity in plasma was measured with a clotting assay (Staclot,Diagnostica Stago, Asnières, France) and the elimination curve wasconstructed. The improved pharmacokinetic of the rFVIIa-trimer-PSAconjugate is illustrated in FIG. 5.

Example 7 Conjugation of rFIX with PSA Using the MAL-FMS-OSU Linker

To 0.6 ml of a solution of recombinant FIX (8 mg/ml) in 20 mM Hepesbuffer, pH 7.4 the bifunctional linker MAL-FMS-OSU (prepared as outlinedby Tsubery et al., J Biol. Chem. 279, 38118-38124 (2004)) was added(concentration: 0.07 mg/mg protein) and incubated at R.T. for 30 min.Derivatized PSA containing a terminal SH group was prepared according toExample 1. The PSA derivative was added to the mixture (concentration:32 mg PSA-SH/mg protein—100 fold molar excess) and incubated foradditional 2 hours at R.T. The reaction was stopped by adding an aqueoussolution of 0.1 M glycine (final concentration 10 mM) and 5 mM cysteine(end concentration 0.5 mM). The free reagents were separated from therFIX-PSA conjugate by Hydrophobic Interaction Chromatography using aprepacked Butyl Sepharose column (HiTrap Butyl FF 5 ml, GE Healthcare).A buffer containing 5 M NaCl (50 mM Hepes-buffer, 5M NaCl, 0.01% Tween80, 6.7 mM CaCl₂, pH 6.9) was added to the PSA-rFIX containing solutionto give a final concentration of 3M NaCl. This mixture was applied tothe column, which was subsequently washed with 10 CV equilibrationbuffer (50 mM Hepes-buffer, 3M NaCl, 0.01% Tween 80, 6.7 mM CaCl₂, pH6.9) and the elution of the rFIX-PSA conjugate was carried out with 50mM Hepes-buffer, pH 7.4, containing 6.7 mM CaCl₂. After elution of theconjugate the pH was adjusted to pH 6.9. The eluate contained 0.24 mg/mlprotein as measured by the BCA-assay, the evidence of bound PSA in theconjugate was proven by the resorcinol assay (Svennerholm, BiochimBiophys Acta 24, 604-611 (1957)). In a final step the eluate wasconcentrated 10 fold by ultrafiltration/diafiltration (UF/DF) using a 30kD membrane (regenerated cellulose/Millipore) against 20 mM Hepes, 50 mMNaCl, 1 mM CaCl₂, pH 7.4.

Example 8 Conjugation of rFVIII with PSA Using the MAL-FMS-OSU Linker

For the preparation of rFVIII-PSA conjugate 6 ml of a solution ofrecombinant FVIII (4.5 mg/ml), derived from the Advate manufacturingprocess, in 20 mM Hepes buffer, pH 7.4 the bifunctional linkerMAL-FMS-OSU (prepared as outlined by Tsubery et al., J Biol. Chem. 279,38118-38124 (2004)) was added (concentration: 0.315 mg/mg protein) andincubated at R.T. for 30 min. Derivatized PSA containing a terminal SHgroup was prepared according to Example 1. The PSA derivative was addedto the mixture (concentration: 27.8 mg PSA-SH/mg protein—450 fold molarexcess) and incubated for additional 2 hours at R.T. The reaction wasstopped by adding an aqueous solution of 0.1 M glycine (finalconcentration 10 mM) and 5 mM cysteine (end concentration 0.5 mM). Thefree reagents were separated from the rFVIII-PSA conjugate byHydrophobic Interaction Chromatography using a prepacked Butyl Sepharosecolumn (HiTrap Butyl FF 5 ml, GE Healthcare). A buffer containing 5 MNaCl (50 mM Hepes-buffer, 5M NaCl, 0.01% Tween 80, 6.7 mM CaCl₂, pH 6.9)was added to the PSA-rFVIII containing solution to give a finalconcentration of 3M NaCl. This mixture is applied to the column, whichwas subsequently washed with 10 CV equilibration buffer (50 mMHepes-buffer, 3M NaCl, 0.1% Tween 80, 5 mM CaCl₂, pH 6.9) and theelution of the rFVIII-PSA conjugate was carried out with Citrate buffer,pH 7.4 (13.6 mM Na₃Citrate, 20 mM CaCl₂, 20 mM Histidine, 0.01% Tween80). After elution of the conjugate the pH was adjusted to pH 6.9. Theeluate contained 2.5 mg/ml protein (BCA assay).

1.-9. (canceled)
 10. A process for the preparation of a compound offormula (2)

said process comprising the step of subjecting a thiol-derivatizedsemi-synthetic biopolymer (HS-POLYMER) and an intermediate compoundhaving a formula

to a coupling reaction to form the compound of formula (2), whereinPOLYMER is a semi-synthetic biopolymer; R¹ is at each occurrenceindependently a (C₁-C₈)-alkyl; R² is independently selected from thegroup consisting of —C(O)NR—, —C(O)NR—(C₁-C₈)-alkyl-NR—, —NRC(O)— and—NRC(O)—(C₁-C₈)-alkyl-NR, R is independently either hydrogen or(C₁-C₈-alkyl; X is —SO₃—R³; R³ is independently selected from the groupconsisting of hydrogen, (C₁-C₈-alkyl and (C₁-C₈)-alkyl-R⁴; R⁴ is apolymer, n is an integer selected from 0, 1, 2, 3, or 4; and m is aninteger selected from 1, 2, 3, or
 4. 11. The process according to claim10, wherein HS-POLYMER is


12. The process according to claim 10, wherein R² is either —C(O)NR— or—NRC(O)— and wherein R is independently either hydrogen or C₁-C₈-alkyl.13. The process according to claim 10, comprising reacting the compoundof formula (2) and a protein or peptide drug which contains one or moregroups for displacement.
 14. (canceled)
 15. The process according toclaim 13, wherein the protein or peptide drug is selected from the groupconsisting of Factor VII, von Willebrand Factor (VWF), Factor VIIa, andFactor IX.